Stable liquid formulations of antibodies

ABSTRACT

The present invention provides stable liquid formulations of antibodies suitable for parenteral administration. Also, provided are aqueous solutions which have high concentrations of therapeutical antibodies which may be used to produce therapeutical liquid formulations. The present invention also relates to uses, such as medical uses, of the stable liquid formulations and processes for the production of the stable liquid formulations.

This application is a continuation of U.S. application Ser. No.10/478,630, filed Apr. 20, 2004, which is a 371 of PCT/EP02/06016, filedMay 31, 2002.

PARTIES TO A JOINT RESEARCH AGREEMENT

Certain subject matter herein was developed during the course of a jointresearch agreement between Novartis Pharma AG and Genentech, Inc.

FIELD OF THE INVENTION

The present invention relates to aqueous solutions which have highconcentrations of therapeutical antibodies and to stable liquidformulations which are based on such aqueous solutions of antibodies.The present invention also relates to uses, such as medical uses, of thestable liquid formulations and processes for the production of thestable liquid formulations.

BACKGROUND OF THE INVENTION

Stable liquid formulations of antibodies are useful for parenteraladministration, such as intravenous (i.v.), intramuscular (i.m.) orsubcutaneous (s.c.) administration. Such formulations must fulfill twokey requirements: 1) the required drug concentration must be achieved,and, 2) the drug must be chemically and physically stable in order tohave a sufficient shelf-life.

For a protein to remain biologically active, a formulation must preserveintact the Conformational integrity and at the same time the protein'smultiple functional groups must be protected from degradation.Degradation pathways for proteins can involve chemical instability orphysical instability. For example, chemical instability can result fromdeamidation, hydrolysis oxidation, beta-elimination or disulfideexchange, while physical instability can result from denaturation,aggregation, precipitation or adsorption, for example. Aggregation isone of the most common protein degradation pathways.

Most current stable formulations of antibodies are not liquidformulations. For example, WO97/04801 describes a stable lyophilizedformulation of anti-IgE antibodies. The stability of proteins in aqueousformulations is of general importance to the pharmaceutical industry.The problem has been addressed by drying the protein, for example, bythe method of freeze-drying. For a patient who needs daily injections ofan antibody, it is of importance that the product is easy to handle, todose and inject. Because a dried antibody formulation is thendistributed and stored in dried form, the patient or medicalprofessional has to reconstitute the dried powder in a solvent beforeuse, which is an inconvenience for the patient.

Thus, it is advantageous to provide a liquid antibody formulation forwhich reconstitution before use is not required.

Furthermore, the freeze-drying process is a costly and time consumingprocess, and it would be advantageous if this step could be avoided whenpreparing a commercial antibody formulation.

It would also be advantage for the manufacture and formulation of atherapeutical product if the final pharmaceutical solution containedonly few or no additives.

Thus, there is a demand on the market for stable, liquid, injectableantibody formulations; and, in particular, for highly concentratedstable, liquid, injectable antibody formulations.

There is also a need for stable aqueous solutions comprising a highconcentration of antibody protein that can be used as a startingmaterial or intermediate in process to obtain stable liquid antibodyformulations of the invention.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a stable aqueous solution comprising anantibody at a concentration of at least 50 mg/ml, and further comprisingat least one acidic component.

Further, there is provided a suitable delivery system which contains theaqueous solution.

Further provided are the uses of the aqueous solution in a nasal sprayor a slow release formulation.

Also provided is the use of the aqueous solution in a drying orfreeze-drying process.

Stable aqueous solution are provided which can be used as anintermediate for the formulation of therapeutical formulations, e.g.further pharmaceutically acceptable components can be added to theaqueous solution in order to obtain the final therapeutical formulation.However, the stable aqueous solution of the invention can itself be usedas a therapeutical formulation; i.e. including no or only few furtheradditives.

Further components which may be added to the stable aqueous solution ofthe invention can be mere pharmaceutical additives which are nottherapeutically active, or they can be therapeutically activesubstances. Also, by-products may or may not be present in the aqueoussolutions of the invention. Accordingly, the stable aqueous solutions ofthe invention may either comprise, consist essentially of, or consist ofan antibody at a concentration of at least 50 mg/ml and at least oneacidic component.

Processes of making a therapeutical formulation employing the aqueoussolution of the invention are also provided.

Thus, in one aspect of the invention a process is provided for thepreparation of a therapeutical liquid formulation comprising anantibody, wherein in a first step an aqueous solution including anantibody at a concentration of at least 50 mg/ml and at least one acidiccomponent is prepared; and, in a second step, at least onepharmaceutically acceptable additive is added to said aqueous solution.

Furthermore, a process is provided for the preparation of atherapeutical liquid formulation comprising an antibody at aconcentration of more than 50 mg/ml, wherein in a first step an antibodysolution in a suitable buffer is concentrated to between about 10 mg/mland about 50 mg/ml; in a second step, the concentrated solution obtainedin the first step is diafiltered with an aqueous solution of at leastone acidic component, optionally containing MgCl₂ and/or CaCl₂ and/orfurther suitable additives; and, in a third step, the solution obtainedin the second step is further concentrated to a concentration of morethan 50 mg/ml.

Also provided is a process for the preparation of a therapeutical liquidformulation comprising an antibody at a concentration of more than 50mg/ml, wherein

-   -   in a first step an antibody solution in a suitable buffer is        concentrated to a concentration of between about 10 mg/ml and        about 50 mg/ml;    -   in a second step, the concentrated solution obtained in the        first step is diafiltered with an aqueous solution of at least        one acidic component;    -   in a third step, the solution obtained in the second step is        further concentrated to an intermediate concentration of between        about 100 and 200 mg/ml, preferably between about 100 and 150        mg/ml;    -   in a fourth step, the intermediate concentrated solution        obtained in the third step is diafiltered with an aqueous        solution of at least one acidic component and further containing        MgCl₂ and/or CaCl₂ and/or further suitable additives,    -   in a fifth step, the solution obtained in the fourth step is        further concentrated to a concentration of more than 150 mg/ml.

DETAILED DESCRIPTION OF THE INVENTION I. High Concentration AqueousSolution of Antibody and Liquid Formulations

The present invention provides highly concentrated aqueous solutions ofantibody and liquid formulations based thereon. The concentrated aqueoussolutions of the invention include a therapeutical antibody and at leastone acidic component. The aqueous solutions therefore generally have apH below pH 7.0. They may or may not include further salts or additives.They may be used as an intermediate in a process to obtain atherapeutical liquid formulation of the invention, but they also may besuitable therapeutical liquid formulations themselves, i.e. without theaddition of further pharmaceutically acceptable additives.

In one aspect the invention provides a stable aqueous solutioncomprising an antibody at a concentration of at least 50 mg/ml, andfurther comprising at least one acidic component. Preferred areconcentrations of the antibody of at least 80 mg/ml, 100 mg/ml, 140mg/ml, 160 mg/ml, 180 mg/ml, 200 mg/ml, 220 mg/ml, 250 mg/ml or even 300mg/ml.

In developing a high concentration stable aqueous solution of antibody,the high viscosity of protein solutions has been identified as a majorobstacle. For example, in physiological saline conditions or buffers atconcentrations above 50 mg/ml antibody solutions, such as for examplesolutions of monoclonal antibody E25, can start to become viscous and/orturbid. The viscosity increases with protein concentration. The highviscosity of antibody solutions is a disadvantage from a medical pointof view as, for example, reconstitution times may be as long as 30 minfor an antibody lyophilizate. Further, after reconstitution andinjection of a dry formulation about 30% of an antibody may be left inthe vial and in the syringe, which severely increases the treatmentcost.

The present invention now provides means to obtain a stable liquidpharmaceutical formulation comprising antibodies, such as anti-IgEantibodies, with a high protein concentration and a low viscosity.

Although we do not wish to be limited by any theoretical speculation,one phenomenon that may contribute to the observed viscosity of aqueousantibody solutions is the self-association of the antibody, or“aggregation”. Antibody aggregates can be soluble or insoluble and bothforms of aggregates can be covalent or non-covalent. The aggregates cangive opalescent solutions, but there can also be non-visible aggregationwhich only can be shown chemically.

In addition to increasing viscosity, aggregation can be detrimental inseveral ways. For example, covalent aggregation in protein formulationsmay be essentially irreversible and could result in the production ofinactive species, which in addition also may be immunogenic.Non-covalent aggregation can lead to loss of activity due toprecipitation.

A “stable” aqueous solution or liquid formulation within the meaning ofthe invention is one in which the antibody therein essentially retainsits physical and chemical stability and integrity upon storage. Variousanalytical techniques for measuring protein stability are available inthe art. Stability can be measured at a selected temperature for aselected time period. For rapid screening, a formulation may be kept at40° C. for 2 weeks to 1 month, at which time stability is measured.Where the formulation is to be stored at 2-8° C., generally theformulation should be stable at 30° C. or 40° C. for at least 1 monthand/or stable at 2-8° C. for at least 1 year. For example, in onepreferred embodiment the aqueous solution of the invention has astability of at least 1 year at about 4° C. The extent of viscosityand/or aggregation can be used as an indicator of protein stability. Forexample, a “stable” formulation may be one wherein less than about 10%and, preferably, less than about 5%, preferably less than about 2%, oreven less than about 1% of the protein is present as an aggregate in theformulation. Aggregation can, for example, be measured by size exclusionchromatography.

The solutions of the invention are stable not only with regard toaggregation but also with regard to the chemical stability of theantibody. Chemical stability may, for example, be measured byhydrophobic interaction chromatography (HIC), for example by HIC-HPLCafter papain digestion. For example, after storage of at least 1 year atabout 4° C. the peak representing unmodified antibody in HIC-HPLC afterpapain digestion decreases no more than 20%, preferably no more than10%, more preferably no more than 5% or even no more than 1%, ascompared to the antibody solution prior to storage.

As the person skilled in the art will readily appreciate, there areother methods suitable to measure the stability of the solutions of theinvention. For example, chemical stability may also be measured bycapillary electrophoresis.

Chemical instability can impair the activity of the antibody inquestion. Examples of chemical instability are degradation of theantibody or changes in tertiary and/or quaternary structure of antibodymolecules. In preferred embodiments the solutions and formulations ofthe invention lose less than 50%, preferably less than 30%, preferablyless than 20%, more preferably less than 10% or even less than 5% or 1%of the antibody activity within 1 year storage under suitable conditionsat about 4° C. The activity of an antibody can be determined by asuitable antigen-binding assay for the respective antibody.

The ability of an acidic component to produce a stable liquid antibodysolution at high protein concentration can be determined by making up asolution including the acidic component to be tested and storing it for24 hours at 22° C. For example, if after this time the solution remainsclear the acidic component has stabilized the antibody and is onesuitable for the use in an aqueous solution according to the presentinvention.

The degree of stability achieved depends on the acid used and on itsconcentration, the antibody concentration, and on the storagetemperature. In general, the higher the concentration of the antibodyand the higher the storage temperature, the shorter the time beforeaggregation occurs. In general higher antibody concentrations requirehigher concentrations of the acidic component.

Accordingly, it is found in the present invention that stable aqueoussolutions and liquid formulations including antibodies having anacceptable viscosity for therapeutical applications can be made in thepresence of specific acidic components.

Preferably, the viscosity of the aqueous solution or liquid formulationof the invention is below 200 mPa·s, preferably below 100 mPa·s,preferably below 70 mPa·s, more preferably below 50 mPa·s, morepreferably below 20 mPa·s or even below 10 mPa·s at a shear rate ofγ=100 (1/s). Another suitable shear rate to measure viscosity ofantibody solutions is γ=220 (1/s).

Such reduced viscosity allows for a aqueous solution or liquidformulation of the invention having a higher concentration of therespective antibody. Thus, advantageously, the same amount of antibodymay be administered in a smaller volume. Also, such smaller volume,advantageously, may allow to produce pre-filled delivery devices thatinclude the entire therapeutical dosage of the respective antibody.Also, if small volumes can be used, a liquid formulation need notnecessarily be isotonic to avoid pain to the patient. However, in onepreferred embodiment the aqueous solution of the invention is isotonic.By “isotonic” it is meant that the formulation of interest hasessentially the same osmotic pressure as human blood. Isotonicformulations will generally have an osmotic pressure from about 250 to350 mOsm. Isotonicity can be measured using a vapor pressure orice-freezing type osmometer, for example.

According to the present invention the acidic component and the amountof acid being used is so chosen as to achieve the desired viscosity andstability of the high concentrated protein solution. Suitable acids thatmay be chosen include organic and inorganic acids. Organic acids of theinvention may be carboxylic acids, such as monocarboxylic, dicarboxylic,tricarboxylic, tetracarboxylic, hydrocarboxylic acids or phenols. Weakorganic acids are preferred acids of the present invention, for examplemonocarboxylic organic acids having a pK-value between 3.0 and 6.0,preferably between 4.5 and 5.0. Preferred examples of acidic componentsof the invention are acetic acid, citric acid, oxalic acid, succinicacid, tartaric acid, lactic acid, malic acid, glycolic acid and fumaricacid. In a particularly preferred embodiment the acidic componentincluded in the aqueous solution is acetic acid.

Preferably, the pH of said aqueous solution or liquid formulation isabove pH 3, for example between pH 3 and pH 7, more preferably it isbetween pH 3 and pH 6, more preferably between pH 4 and pH6, or evenbetween pH 5 and pH 6. In one preferred embodiment the pH is about pH5.0 or about pH 6.0. Certain pH ranges are particularly preferred, forexample, preferred is a pH below pH 6.0, or below pH 5.8, or below pH5.6 or below pH 5.4, and a pH that is above pH 4.0, or above pH 4.2, orabove pH 4.4, or above pH 4.6 or above pH 4.8, or above pH 5.0.

Preferably the acidic component of the invention, such as acetic acid,is present in a final concentration of at least 0.001%, preferably atleast 0.01%, more preferably between 0.01%-0.2%. In one embodiment ofthe invention no additional buffering agent is present in the aqueoussolution or liquid formulation of the invention. In another embodimentof the invention no sodium salt, such as for example sodium acetate ispresent in the aqueous solution or liquid formulation of the invention.

The concentration of the antibody, such as an anti-IgE antibody such asfor example E25 (as defined hereinbelow), is above 50 mg/ml, for exampleit may be between 100 and 200 mg/ml and can go up to 300 mg/ml.Preferred is a concentration of at least 80, 100, 140, 160, 180, 200,220, 250 or even 300 mg/ml. One preferred range is between 100 and 220mg/ml for injectable solutions. If a protein shall be delivered via thenasal or even the oral route, preferred concentrations are at least 250mg/ml or even 300 mg/ml, as high concentrations are particularlydesirable for the delivery via the nasal or oral route.

The aqueous solution or liquid formulation of the invention may alsocontain more than one antibody as necessary for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect the other antibody. The aqueous solution orliquid formulation herein may also include an additional therapeuticalprotein which is not an antibody. Such antibodies or proteins aresuitably present in combination in amounts that are effective for thepurpose intended. When including a further protein component in theaqueous solution, the total protein concentration should be taken intoaccount when choosing the concentration of the acidic component.

In one aspect the present invention also provides for a stable aqueoussolution consisting merely of an antibody at a concentration of at least50 mg/ml and an acidic component. In another aspect the stable aqueoussolution however may also consist essentially of an antibody at aconcentration of at least 50 mg/ml and an acidic component, inparticular it may further include by-product or therapeutically inactiveadditives.

Preferably, the aqueous solution or liquid formulation of the inventionfurther includes CaCl₂ and/or MgCl₂. In a preferred embodiment theconcentration of CaCl₂ is within the range of 50-200 mM, more preferablywithin 50-130 mM, preferably 100-130 mM, most preferably about 100 mM.In another preferred embodiment the concentration of MgCl₂ is within therange of 50-200 mM, more preferably within 50-130 mM, preferably 100-130mM, most preferably about 100 mM. Stable aqueous solutions or liquidformulations including MgCl₂ are a particularly preferred embodiment ofthe present invention. In a further preferred embodiments these aqueoussolutions or liquid formulations further include a detergent and/or asugar.

II. Antibodies

The term “antibody” is used in a broad sense. The term “antibody”specifically covers monoclonal antibodies (including full lengthantibodies which have an immunoglobulin Fc region), antibodycompositions with polyepitopic specificity, bispecific antibodies,diabodies, and single-chain molecules, as well as antibody fragmentsand/or derivatives such as, for example, Fab, F(ab′)₂, and Fv fragmentsor other antigen-binding fragments. For example, an antibody derivativemay be a PEGylated form of an antibody or antibody fragment.

In a preferred embodiment the antibody used in the aqueous solution ofthe invention has an isoelectric point between pH 6 and pH 8.

The term “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod or may be made by recombinant DNA methods. The “monoclonalantibodies” may also be isolated from phage antibody libraries.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity.

“Humanized” forms of non-human antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof, such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies, whichcontain minimal sequence derived from non-human immunoglobulin. Usually,humanized antibodies are human immunoglobulins in which residues from acomplementarity determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species. In some instances, Fvframework region residues of the human immunoglobulin are replaced bycorresponding non-human residues. Also, complementarity determiningregion (CDR) residues originating from the non-human species may bereplaced by corresponding human residues. Furthermore, humanizedantibodies may comprise residues which are found neither in therecipient antibody nor in the imported CDR or framework sequences.

In a particularly preferred embodiment the antibody or antibodyderivative is selected from anti-IgE antibodies, such as E25, E26, E27(described in WO99/01556 as rhuMAbE-25, rhuMAbE-26, and rhuMAbE-27,respectively) or their fragments and derivatives. Preferably theanti-IgE antibody is a humanized murine antibody or a fully humanantibody. Most preferably the anti-IgE antibody is Omalizumab, which isalso named “E25”. Another preferred anti-IgE antibody is named “E26” asfurther defined hereinbelow.

Generally, anti-IgE antibodies are described in the prior art, and ingreater detail in the International applications WO 93/04173 and WO99/01556. For example, WO 99/01556 specifically describes Omalizumab,also named E25, in FIG. 12, and in the sequences ID-No. 13-14. Antibodymolecules comprising a E26 sequence are described in WO 99/01556 and areselected from the group of F(ab) fragment (Sequence ID Nos. 19-20), sFvfragment (Sequence ID No. 22) and F(ab)′₂ fragment (Sequence Nos.24-25), in accordance to FIGS. 12-15. Within this invention, the termsE25 and E26 shall be construed accordingly. Preferably, the IgEantibodies of the instant invention do not result in histamine releasefrom mast cells or basophils.

Furthermore, U.S. Pat. No. 5,449,760 generally describes anti-IgEantibodies that bind soluble IgE but not IgE on the surface of B cellsor basophils. Antibodies such as these bind to soluble IgE and inhibitIgE activity by, for example, blocking the IgE receptor binding site, byblocking the antigen binding site and/or by simply removing the IgE fromcirculation. Additional anti-IgE antibodies and IgE-binding fragmentsderived from the anti-IgE antibodies are described in U.S. Pat. No.5,656,273. U.S. Pat. No. 5,543,144 describes further anti-IgE antibodiesthat are suitable for this invention, in particular anti-IgE antibodiesthat bind soluble IgE and membrane-bound IgE on IgE-expressing B cellsbut not to IgE bound to basophils.

III. Aqueous Antibody Solutions Including Suitable Additives LiquidFormulations

It has been surprisingly found that after the preparation of thehighly-concentrated aqueous antibody acid solution according to theinvention different ingredients can be added without a substantialincrease in viscosity. The antibody acid solution can for example bemixed with sugars, detergents and/or other additives. Accordingly thepresent invention also describes methods suitable for the preparation oflong-term stable liquid formulations of antibodies including suchadditives. Also provided are the aqueous solutions including suchadditives themselves.

A person skilled in the art will appreciate that a wide variety ofexcipients may be used as additives. Components that may be used asadditives are e.g.:

a) liquid solvents, co-solvents, e.g. an alcohol, e.g. isopropanol,

b) sugars or a sugar alcohols, e.g. mannitol, trehalose, sucrose,sorbitol, fructose, maltose, lactose or dextrans,

c) detergents, e.g. TWEEN 20, 60 or 80 (polysorbate 20, 60 Or 80)

d) buffering agents, e.g. acetate buffer

e) preservatives, e.g. benzalkonium chloride, benzethonium chloride,tertiary ammonium salts and chlorhexidinediacetate.

f) isotoning agents, e.g. sodium chloride

g) carriers, e.g. polyethylene glycol (PEG), recombinant human serumalbumin

h) antioxidants e.g. ascorbic acid and methionine

i) chelating agents e.g. EDTA

j) biodegradable polymers e.g. polyesters

k) salt-forming counterions e.g. sodium

A “preservative” within the meaning of the invention is a compound whichcan be added to the diluent to essentially reduce bacterial action inthe reconstituted formulation, thus facilitating the production of amulti-use reconstituted formulation, for example. For example,preservatives may advantageously be included in solutions suitable fornasal administration or in solutions for use with multiple peninjectors.

Preferred compounds to be added as further additives are detergents suchas TWEEN 20, sugars such as sucrose, fructose, mannitol andpreservatives. Preferably, additives derived from animal origin such asgelatine or serum albumin (e.g. BSA) are excluded from formulations ofthe invention. Please replace the second full paragraph of page 13 withthe following paragraph:

Generally, acceptable additives are nontoxic to recipients at thedosages and concentrations employed. The formulations to be used for invivo administration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes, alternatively,sterility of the entire mixture may be accomplished by autoclaving theingredients, except for protein, at about 120° C. for about 30 minutes,for example.

The percentage of the acid solution and the amount of additives used canvary and depends on the intended use. For example during differentmanufacturing steps the concentration of the acid solution can differfrom the concentration of the final product.

It should be noted that certain additives such as ethanol, phosphatebuffer saline (PBS), or citrate buffer, may induce gelation, increasedviscosity and/or aggregation of the antibody in question under certainpH conditions. If the problems cannot be avoided by routine changes inpH, such additives should preferably not be used for preparingcompositions of this invention.

A liquid formulation may, for example, be made by adding the additivesto an aqueous solution of the antibody and then stirring to dissolve.Any suitable stirrer may be used, e.g. a vortex mixer. It is preferredto dissolve the antibody in an aqueous solution of the acid and then toadd an aqueous solution of the additives. The stirring may preferably becarried out under an inert gas atmosphere, such as nitrogen or argon,and the resulting solution may preferably be degassed under vacuum. Theinert gas atmosphere and degassing both may help to prolong thestability of the solution. After preparation the solution may be storedin glass or plastics containers.

Preferably, the aqueous solution or liquid formulation of the inventionfurther includes CaCl₂ and/or MgCl₂. In a preferred embodiment theconcentration of CaCl₂ is within the range of 50-200 mM, more preferably50-130 mM, preferably 100-130 mM, most preferably about 100 mM. Inanother preferred embodiment the concentration of MgCl₂ is within therange of 50-200 mM, more preferably 50-130 mM, preferably 100-130 mM,most preferably about 100 mM.

In one preferred embodiment the aqueous solution or liquid formulationof the invention further includes a detergent, such as for example TWEEN20, TWEEN 60 or TWEEN 80.

In another preferred embodiment the aqueous solution or liquidformulation of the invention further includes at least one sugar. In afurther preferred embodiment the aqueous solution or liquid formulationof the invention further includes at least one sugar selected from thegroup comprising trehalose, sucrose, mannitol, sorbitol, fructose,maltose, lactose or a dextran. However, in one embodiment of theinvention the aqueous solution or liquid formulation of the inventiondoes not include maltose.

In another embodiment the aqueous solution or liquid formulation of theinvention further includes at least one buffering agent.

One desirable anti-IgE antibody aqueous solution discovered hereinincludes an anti-IgE antibody in amount between 100 and 200 mg/ml,preferably of about 190 mg/ml or of about 220 mg/ml, and CaCl₂ or MgCl₂in an amount between 50 and 200 mM, preferably of about 50 mM or ofabout 100 mM, optionally a buffer and optionally a detergent, such as aTween 20, e.g. at a concentration of about 0.02%. Preferably, thisanti-IgE formulation is stable at 8° C. for at least 1 year.

IV. Devices

The aqueous solution or liquid formulation of the invention may, forexample, be used with standard ampoules, vials, pre-filled syringes ormultiple administration systems. In preferred embodiments, the aqueoussolution may be administered to the patient by subcutaneousadministration. For example, for such purposes, the formulation may beinjected using a syringe. However, other injection devices foradministration of the formulation are available such as injector pens,and subcutaneous patch delivery systems such as, for example, chipdevices. However, the aqueous solution may also be administered to thepatient by inhalation devices. Conventional systems for delivery ofmolecules through the nasal passages and the lung include metered doseinhalers, and liquid jet and ultrasonic nebulizers.

Accordingly, in one aspect the present invention also provides adelivery system which contains the aqueous solution selected from thegroup of single use injection syringes or inhalation devices.

The delivery system comprises a container. Suitable containers include,for example, bottles, vials (e.g. dual chamber vials), syringes (such asdual chamber syringes) and test tubes. The container may be formed froma variety of materials such as glass or plastic. The container holds theaqueous solution and the label on, or associated with, the container mayindicate directions for use. The label may for example indicate that theaqueous solution is useful or intended for subcutaneous administration.The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g. from 2-6 administrations) of theaqueous solution.

Accordingly, also provided is the use of the aqueous solution or liquidformulation according to the invention for the production of a deliverysystem for the use treatment of a disease.

In another embodiment of the invention, an article of manufacture isprovided which contains the aqueous solution of the present inventionand provides instructions for its use. Thus, an article of manufactureis provided herein which comprises:

a) container which holds a concentrated aqueous solution of an antibody;and

b) instructions for diluting the concentrated aqueous solution with adiluent to a protein concentration in the diluted formulation of atleast about 50 mg/mL. The article of manufacture may further comprise asecond container which holds a diluent (eg. bacteriostatic water forinjection comprising an aromatic alcohol).

The article of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

V. Specific Formulations

In another aspect of the invention there is provided a slow releaseformulation comprising the aqueous solution or liquid formulation of theinvention. Preferred is a slow release formulation selected from thegroup of polymeric nano or microparticles, or from gels.

In a particularly preferred embodiment the slow release formulation is agel such as a hyaluronic acid gel.

Besides convenience, slow release formulations offer other advantagesfor delivery of protein drugs including protecting the protein over anextended period from degradation or elimination, and the ability todeliver the protein locally to a particular site or body compartmentthereby lowering overall systemic exposure.

The present invention, for example, also contemplates injectable depotformulations in which the protein is embedded in a biodegradablepolymeric matrix. Polymers that may be used are the homo- andco-polymers of lactic and glycolic acid (PLGA). PLGA degrades byhydrolysis to ultimately give the acid monomers and is chemicallyunreactive under the conditions used to prepare, for example,microspheres and thus does not modify the protein. After subcutaneous orintramuscular injection, the protein is released by a combination ofdiffusion and polymer degradation. By using polymers of differentcomposition and molecular weight, the hydrolysis rate can be variedthereby allowing release to last from days to months.

In a further aspect the present invention provides a nasal spraycomprising the aqueous solution or liquid formulation of the presentinvention.

VI. Uses and Processes for Preparation

In a further aspect of the invention the use of an acidic component forthe preparation of an aqueous solution comprising an antibody having aconcentration of at least 50 mg/ml is provided.

Also provided is a process for the preparation of a aqueous solutionaccording to the invention, which process comprises admixing an antibodywith an acidic component.

Also provided is a process for the preparation of a therapeutical liquidformulation comprising an antibody, wherein in a first step an aqueoussolution including an antibody at a concentration of at least 50 mg/mland at least one acidic component is prepared, and, in a second step, atleast one pharmaceutically acceptable additive is added to said aqueoussolution.

Also provided is a process for the preparation of a therapeuticalformulation including an antibody, which process comprises adding anacidic component on the last purification step of the preparation ofsaid antibody. Such last step may, for example, be an elution step, abuffer exchange step or a step comprising continuous diafiltration.

Furthermore, a process is provided for the preparation of atherapeutical liquid formulation comprising an antibody at aconcentration of more than 50 mg/ml, wherein in a first step an antibodysolution in a suitable buffer is concentrated to a concentration betweenabout 10 mg/ml and about 50 mg/ml; in a second step, the concentratedsolution obtained in the first step is diafiltered with an aqueoussolution of at least one acidic component, optionally containing MgCl₂and/or CaCl₂ and/or further suitable additives; and, in a third step,the solution obtained in the second step is further concentrated to aconcentration of more than 50 mg/ml.

For example, the aqueous solution of at least one acidic component maybe a solution of acetic acid, such as a solution of between about 0.01%and about 0.1% acetic acid. MgCl₂ and/or CaCl₂ may be present at aconcentration within the range of 50-200 mM, preferably 50-130 mM, morepreferably 100-130 mM, most preferably about 100 mM. In a furtherpreferred embodiments these aqueous solutions further include adetergent and/or a sugar.

Also provided is a process for the preparation of a therapeutical liquidformulation comprising an antibody at a concentration of more than 50mg/ml, wherein

-   -   in a first step an antibody solution in a suitable buffer is        concentrated to a concentration of between about 10 mg/ml and        about 50 mg/ml;    -   in a second step, the concentrated solution obtained in the        first step is diafiltered with an aqueous solution of at least        one acidic component;    -   in a third step, the solution obtained in the second step is        further concentrated to an intermediate concentration of between        about 100 and 200 mg/ml, preferably between about 100 and 150        mg/ml;    -   in a fourth step, the intermediate concentrated solution        obtained in the third step is diafiltered with an aqueous        solution of at least one acidic component containing MgCl₂        and/or CaCl₂ and/or further suitable additives; and,    -   in a fifth step, the solution obtained in the fourth step is        further concentrated to a concentration of more than 150 mg/ml.

The diafiltration is generally carried out at constant retentate volume,with at least 5 volumes, or preferably 8 volumes, of diafiltrationbuffer.

In a preferred embodiment a solution of MgCl₂ and/or CaCl₂ and/orfurther suitable additives may directly be added to the intermediateconcentrated solution obtained in the third step of the above 5-stepprocess. If MgCl₂ and/or CaCl₂ and/or further suitable additives aredirectly added, the fourth step (i.e. diafiltration with an aqueoussolution of at least one acidic component containing MgCl₂ and/or CaCl₂and/or further suitable additives) thereafter may be omitted if nofurther adjustment of the respective concentrations of the salts and/oradditives is required. Generally, the 5-step process of the inventionwhich adds the salts and/or additives only to an intermediateconcentrated solution of antibody avoids the appearance of aggregatesand/or turbidity in solutions of the process.

In one preferred embodiment, in the fourth step a concentrated aqueoussolution of MgCl₂ (or CaCl₂), for example at concentration 1 M, is addeddirectly into an ultrafiltration system, to give approximately thedesired resulting concentration (for example 50 mM or 100 mM).

In preferred embodiments of the processes of the invention carboxylicacids, such as acetic acid, are employed as the acidic component. Inpreferred embodiments of these processes no salt of a carboxylic isadded in the process. In particular, in these embodiments it ispreferred if no salt of the corresponding carboxylic acid is added.

VII. Medical Uses

In one aspect, the present invention also provides the aqueous solutionof the invention for use in medicine. In particular, the use of theaqueous solution for the manufacture of a medicament for the treatmentof disease, such as for example an allergic disease, is provided.

The appropriate dosage of the protein will depend, for example, on thecondition to be treated, the severity and course of the condition,whether the protein is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the protein, the type of antibody used, and the discretion of theattending physician. The antibody is suitably administered to thepatient at one time or over a series of treatments and may beadministered to the patient at any time from diagnosis onwards. Theantibody may be administered as the sole treatment or in conjunctionwith other drugs or therapies useful in treating the condition inquestion.

The uses for a formulation including an anti-IgE antibody, for example,include the treatment or prophylaxis of IgE-mediated allergic diseases,parasitic infections, interstitial cystitis and asthma, in particularallergic asthma, allergic rhinitis and atopic dermatitis, for example.Depending on the disease or disorder to be treated, a therapeuticallyeffective amount of the anti-IgE antibody may be administered to thepatient.

In another aspect there is provided the use of the aqueous solution ofthe invention in a drying or freeze-drying process.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention.

EXAMPLES Example 1

Solutions of 40 mg/ml E25 in the production buffer (10 mM histidinebuffer 10% sucrose) were dialyzed against large volumes of water and of0.01% acetic acid. The obtained E25 solutions, in water and in 0.01%acetic acid, were concentrated by filtration. The obtained E25 solutionin water (99 mg/ml E25, pH 7.04) was much more viscous than the 0.01%acetic acid E25 solution (98 mg/ml E25, pH 5.4).

The beneficial effect of acetic acid in obtaining solutions with reducedviscosity was further documented. For example, 160 mg/ml E25 could beeasily obtained in 0.1% acetic acid (final protein solution had a pH of4.8) or solution of 183 mg/ml E25 in 0.01% acetic acid. A water solutionof E25 of 170 mg/ml could also be prepared, but it was much more viscousthan all the acetic acid solutions.

No chemical degradation was detected by capillary zone electrophoresis(CZE) after storing the solutions at 8° C. for 10 days.

Example 2

The buffer of a solution of 40 mg/ml E25 in the production buffer (10 mMhistidine buffer 10% sucrose) was exchanged in a diafiltration equipmentto 0.1% acetic acid. After that the E25 solution was concentrated byultrafiltration to 161 mg/ml. The solution was fluid, no aggregation oropalescence was observed. The recovery was very good, about 95%. Thissolution of 161 mg/ml was further concentrated by filtration throughcentrifugation using Centricone tubes. Fluid, clear solutions of E25 in0.1% acetic acid with concentrations of 214 mg/ml and also 297 mg/mlwere obtained. The solutions can be easily handled through syringeneedles and permit the development of a single use prefilled syringewith small volume (e.g., 0.5 ml to 1 ml).

Example 3

A solution of 40 mg/ml E25 in the final production buffer (containing0.02% TWEEN 20) was dialyzed against 0.1% acetic acid. The resulted E25solution in 0.1% acetic acid (still containing TWEEN 20 detergent) wasconcentrated by filtration through centrifugation using Centricone: aconcentration of 243 mg/ml E25 was reached. The solution fluidity wassimilar to the fluidity of the solutions without TWEEN 20, showing thatthe detergent is compatible with the high protein concentratedformulation.

Example 4

The unexpected beneficial effect of acetic acid can be illustrated inthe following experiment. Solutions of 161 mg/ml E25 in 0.1% (17.5mM)acetic acid (pH 4.8) were dialyzed against i) 17.5 mM phosphatebuffer with 145 mM NaCl (PBS) pH 7.4; ii) 17.5 mM acetate buffer pH 4.8;and iii) 17.5 mM citrate buffer pH 4.8. Unexpectedly, in the citratebuffer pH 4.8 solution E25 aggregated and the solution became whiteturbid. This did not happen in the other solutions. The phosphate bufferwas more viscous than the acetate buffer solution. The phosphate bufferE25 solution became opalescent after one day at room temperature.

Example 5

The viscosity of different E25 solutions was measured. All measurementswere performed with a Paar Physica cone and plate rheometer at 23° C.The results are shown in Table 1 and Table 2 below.

TABLE 1 Viscosity η Viscosity η (mPa · s) at (mPa · s) at shear rate ofshear rate of E25 Samples γ = 100 (1/s) γ = 225 (1/s) Notes 97.4 mg/ml,0.01% acetic acid 22.4 21.2 Beneficial effect of 0.01% acetic acidcompared to water   99 mg/ml, in water 33.9 32.0  222 mg/ml, 0.1% aceticacid 126 123  222 mg/ml, 0.1% acetic acid, 50 mM 66.6 63.2 CaCl2  222mg/ml, 0.1% acetic acid, 100 mM 59.2 55 CaCl₂ decreases the viscosityCaCl2  222 mg/ml, 0.1% acetic acid, 50 mM 79.5 77.2 MgCl2  222 mg/ml,0.1% acetic acid, 100 mM 67.9 64.5 MgCl₂ decreases the viscosity MgCl2 222 mg/ml, 0.1% acetic acid, 50 mM 109 103 NaCl  222 mg/ml, 0.1% aceticacid, 100 mM 114 112 NaCl  222 mg/ml, 0.1% acetic acid, 150 mM 117 118No effect of NaCl NaCl

TABLE 2 Viscosity η (mPa · s) at share rate E25 Samples of γ = 1 Notes222 mg/ml, 0.1% acetic acid 368 222 mg/ml, 0.1% acetic acid, 351 50 mMNaCl 222 mg/ml, 0.1% acetic acid, 1080 100 mM NaCl 222 mg/ml, 0.1%acetic acid, 2140 NaCl increases the viscosity 150 mM NaCl at very lowshear rates

Example 6

A solution of 161 mg/ml in 0.1% acetic acid was lyophilized in a glassvial.

After lyophilization the obtained E25 cake was difficult to solubilizewith 0.1% acetic acid. However, the lyophilized E25 could be solubilizedvery quickly with a reconstitution solution of 0.1% acetic acidcontaining 100 mM CaCl₂. The lyophilized E25 was reconstituted at aconcentration of 235 mg/ml. (the volume of the reconstitution solutionwas smaller than the initial volume of the solution). This example showsthat CaCl₂ has unexpected beneficial effects in solubilizing E25lyophilisates.

Example 7 General Method for the Preparation of High Concentrated LiquidFormulations

The starting solution is a solution of purified antibody at lowconcentration (lower than the high concentrations of the invention) inan aqueous buffer, for example in the buffer resulting from thepreceding process step (for example in the case of E25: 25 mM TRISbuffer pH 8 containing about 200 mM NaCl). The pH of this solution isadjusted to a value below the isoelectric point of the antibody, forexample to pH 5, with an acid, for example with 5% acetic acid. Theresulting solution is then concentrated and diafiltered byultrafiltration, preferably in a tangential-flow filtration system,using a membrane able to retain quantitatively the antibody, for examplewith a cutoff of 30 kD or 10 kD.

In general the following 3-steps procedure applies:

-   -   In a first step, the antibody solution is concentrated to an        intermediate concentration, for example 40 mg/ml. Normally the        retentate obtained is opalescent, due to antibody aggregation.    -   In a second step, the concentrated solution is diafiltered with        an aqueous acetic acid solution (for example 0.01% or 0.1%        acetic acid) containing MgCl₂ or CaCl₂ (for example at        concentration 50 mM or 100 mM) and optionally containing other        additives (for example a sugar). The diafiltration is generally        carried out at constant retentate volume, with at least 5        volumes, or preferably 8 volumes, of diafiltration buffer.        During the diafiltration the antibody solution is turbid.    -   In a third step, the diafiltered solution is further        concentrated to a high concentration, for example higher or        equal to 240 mg/ml. The final turbid retentate is then recovered        out of the ultrafiltration system.

After an optional addition of additives (for example a detergent andeventually other excipients, e.g. sugars, buffering agents) and afterfiltration through a 0.2 μm filter, a high concentrated liquidformulation is obtained, which is clear and stable if stored at about 4°C.

In a preferred embodiment of this general method, in order to processless turbid solutions, the following 5-steps procedure applies:

-   -   In a first step, the antibody solution is concentrated to an        intermediate concentration, for example 40 mg/ml. Normally the        retentate obtained is opalescent, due to antibody aggregation.    -   In a second step, the concentrated solution is diafiltered with        an aqueous solution containing only acetic acid (for example        0.01% or 0.1% acetic acid). The diafiltration is generally        carried out at constant retentate volume, with at least 5        volumes, or preferably 8 volumes, of diafiltration buffer.        Normally, a decrease of the turbidity is observed during the        diafiltration and the solution turns clear.    -   In a third step, the diafiltered solution is further        concentrated to a higher intermediate concentration, preferably        of about 120-130 mg/ml. Then, a concentrated aqueous solution of        MgCl₂ (or CaCl₂), for example at concentration 1 M, is added        directly into the ultrafiltration system, to give approximately        the desired resulting concentration (for example 50 mM or 100        mM). After mixing by retentate recirculation, a decrease of the        retentate pressure is observed, due to the resulting lower        viscosity. The retentate obtained remains clear or slightly        turbid.    -   In a fourth step, the solution is diafiltered with the same        acetic acid solution as used for the first diafiltration (for        example 0.01% or 0.1% acetic acid), but this time containing        additionally MgCl₂ (or CaCl₂) at the desired concentration (for        example 50 mM or 100 mM), in order to adjust exactly this        concentration in the retentate. The diafiltration is generally        carried out at constant retentate volume, with at least 5        volumes, or preferably 8 volumes, of diafiltration buffer.    -   In a fifth step, the diafiltered solution is further        concentrated to a high concentration, for example higher or        equal to 240 mg/ml. The final clear or slightly turbid retentate        is then recovered out of the ultrafiltration system.

After an optional addition of additives (for example a detergent andeventually other excipients, e.g. sugars, buffering agents) and afterfiltration through a 0.2 μm filter, a high concentrated liquidformulation is obtained, which is clear and stable if stored at about 4°C.

Example 8 Preparation and Viscosity of a Formulation Containing AceticAcid and MgCl₂

About 12 ml of the liquid formulation [257 mg/ml E25, 0.1% acetic acid,50 mM MgCl₂] were prepared by ultrafiltration in a tangential-flowfiltration system (membrane area: 150 cm², membrane cutoff: 10 kD, holdup volume of the system: 9 ml, retentate pressure: 2-3 bar), accordingto the 5-steps procedure described in Example 7.

The starting solution was a solution of purified E25 antibody atconcentration 4.8 mg/ml in a 25 mM TRIS buffer pH 8 containing about 200mM NaCl. After pH adjustment to pH 5 with 5% acetic acid, the followingsteps were carried out:

-   -   In a first step, the solution was concentrated to 40 mg/ml.    -   In a second step, the concentrated solution was diafiltered at        constant retentate volume with 8 volumes of 0.1% acetic acid.    -   In a third step, the diafiltered solution was concentrated to        127 mg/ml and, after retentate recirculation during 5 minutes        with the filtrate line closed, a sample was taken for viscosity        measurement. Then, an aqueous solution of 1 M MgCl₂ was added        directly into the ultrafiltration system, to give approximately        a resulting MgCl₂ concentration of 50 mM. After reconcentration        to the initial retentate volume (i.e. the volume before the        addition of MgCl₂) and after retentate recirculation during 3        minutes with the filtrate line closed, a sample of the retentate        was taken for viscosity measurement.    -   In a fourth step, the solution was diafiltered at constant        retentate volume with 8 volumes of 0.1% acetic acid containing        50 mM MgCl₂.    -   In a fifth step, the diafiltered solution was concentrated to        about 260 mg/ml. After recovery of the retentate out of the        ultrafiltration system and filtration through a 0.2 μm filter, a        sample was taken for viscosity measurement. An other sample was        diluted to 200 mg/ml with 0.1% acetic acid containing 50 mM        MgCl₂, also for viscosity measurement.

The viscosity measurements of the samples were performed with a PaarPhysica cone and plate rheometer at 23° C. and at a shear rate of 220s⁻¹. The following results were obtained:

Process step: E25 conc. pH Viscosity in 0.1% acetic acid, before MgCl₂127 mg/ml 4.43 12.1 mPa · s addition: in 0.1% acetic acid, after MgCl₂127 mg/ml 4.48 8.27 mPa · s addition: in 0.1% acetic acid, 50 mM MgCl₂:257 mg/ml 3.84  135 mPa · s in 0.1% acetic acid, 50 mM MgCl₂: 200 mg/ml3.82 37.1 mPa · s

Example 9 Viscosity of E25 Formulations Versus the Acetic AcidConcentration

The same experiment as stated in Example 8 was carried out severaltimes, changing only the acetic acid concentration used for thediafiltration buffers, but keeping the final MgCl₂ concentration equalto 50 mM. The different high concentrated E25 solutions obtained werethen diluted to about 200 mg/ml, using the respective diafiltrationbuffers (i.e. the corresponding acetic acid solutions containing 50 mMMgCl₂), for pH and viscosity measurements.

The viscosity measurements were performed with a Paar Physica cone andplate rheometer at 23° C. and at a shear rate of 220 s⁻¹. The followingresults were obtained:

Formulation buffer: E25 conc. pH Viscosity   0.1% acetic acid, 50 mMMgCl₂: 200 mg/ml 3.82 37.1 mPa · s  0.05% acetic acid, 50 mM MgCl₂: 200mg/ml 4.03 31.4 mPa · s  0.025% acetic acid, 50 mM MgCl₂: 206 mg/ml 4.2633.8 mPa · s  0.01% acetic acid, 50 mM MgCl₂: 195 mg/ml 4.63 38.3 mPa ·s  0.005% acetic acid, 50 mM MgCl₂: 197 mg/ml 4.83 54.5 mPa · s 0.0025%acetic acid, 50 mM MgCl₂: 205 mg/ml 5.01  106 mPa · s  0.001% aceticacid, 50 mM MgCl₂: 201 mg/ml 5.13  115 mPa · s    0% acetic acid, 50 mMMgCl₂: 198 mg/ml 5.35  200 mPa · s

As shown by these results, when lowering the acetic acid concentrationfrom 0.1% to 0% (at constant antibody concentration and at constant.MgCl₂ concentration) the viscosity remains approximately constant in theconcentration range between 0.1% and 0.01%, but increases drastically ifthe concentration is further reduced.

It was found that this “transition concentration” of about 0.0075%acetic acid corresponds to 1.3 mM, which corresponds to the E25 molarconcentration corresponding to 200 mg/ml. Accordingly, in one embodimentof the invention the concentration of the acidic component of theinvention is so chosen as to be about equal or above the molarconcentration of the antibody of the aqueous solution or formulation ofthe invention.

Example 10 Viscosity of Formulations Containing Acetic Acid and EitherMgCl₂ or CaCl₂

About 18 ml of the liquid formulation [237 mg/ml E25, 0.01% acetic acid,50 mM MgCl₂] were prepared by ultrafiltration in a tangential-flowfiltration system (membrane area: 150 cm², membrane cutoff: 10 kD, holdup volume of the system: 10 ml, retentate pressure: 2.5-4 bar),according to the 3-steps procedure described in Example 7:

The starting solution was a solution of purified E25 antibody atconcentration 4.8 mg/ml in a 25 mM TRIS buffer pH 8 containing about 200mM NaCl. After pH adjustment to pH 5 with 5% acetic acid, the followingsteps were carried out:

-   -   In a first step, the solution was concentrated to 40 mg/ml.    -   In a second step, the concentrated solution was diafiltered at        constant retentate volume with 8 volumes of 0.01% acetic acid        containing 50 mM MgCl₂.    -   In a third step, the diafiltered solution was concentrated to        230-240 mg/ml. After recovery of the retentate out of the        ultrafiltration system and filtration through a 0.2 μm filter,        two samples was taken for viscosity measurement (the first one        as is, the second one after addition of 0.02% of TWEEN 20). Two        other samples were diluted to about 210 mg/ml with 0.01% acetic        acid containing 50 mM MgCl₂, also for viscosity measurement (the        first one as is, the second one after addition of 0.02% of TWEEN        20).

The same experiment was repeated, but using CaCl₂ instead of MgCl₂,giving the liquid formulation [233 mg/ml E25, 0.01% acetic acid, 50 mMCaCl₂].

The viscosity measurements were performed with a Paar Physica cone andplate rheometer at 23° C. and at a shear rate of 220 s⁻¹. The followingresults were obtained:

Formulation buffer: E25 conc. Viscosity 0.01% acetic acid, 50 mM MgCl₂:237 mg/ml 83.5 mPa · s 0.01% acetic acid, 50 mM MgCl₂, 0.02% 237 mg/ml86.6 mPa · s Tween 20: 0.01% acetic acid, 50 mM CaCl₂: 233 mg/ml 60.5mPa · s 0.01% acetic acid, 50 mM CaCl₂, 0.02% 233 mg/ml 59.1 mPa · sTween 20: 0.01% acetic acid, 50 mM MgCl₂: 211 mg/ml 40.5 mPa · s 0.01%acetic acid, 50 mM MgCl₂, 0.02% 211 mg/ml 42.1 mPa · s Tween 20: 0.01%acetic acid, 50 mM CaCl₂: 207 mg/ml 34.8 mPa · s 0.01% acetic acid, 50mM CaCl₂, 0.02% 207 mg/ml 31.6 mPa · s Tween 20:

As shown by these results, the viscosity values are slightly lower ifCaCl₂ is used instead of MgCl₂. Moreover, the TWEEN 20 at concentration0.02% has no influence on the viscosity.

Example 11 Preparation and Stability of High Concentrated LiquidFormulations

The three following high concentrated liquid formulations were preparedby ultrafiltration (about 65 ml each, starting with E25 drug substancewithout TWEEN), according to the 5-steps procedure described in Example7:

Formulation # F1 F2 F3 Lot # NVP-IGE025- NVP-IGE025- NVP-IGE025- 01PP0101PP02 01PP03 Composition: E25 196 mg/ml 201 mg/ml 167 mg/ml acetic acid0.1% 0.1% 0.05% MgCl₂ 50 mM 50 mM 50 mM Mg-acetate — 30 mM 45 mMTrehalose 27 mg/ml — — Tween 20 0.02% 0.02% 0.02% pH 4.50 4.95 5.20Tonicity 273 mOsm/kg 252 mOsm/kg 277 mOsm/kg Viscosity 39.9 mPa · s 48.3mPa · s 19.5 mPa · s (at 220 s⁻¹; 23° C.)

These formulations were put on a stability program and were found to bestable after 6-months storage at 5° C. (study ongoing). The followingassays were carried out: SEC (size-exclusion chromatography), HIC(hydrophobic-interaction chromatography after papain-digestion) andBioassay (IgE-Receptor binding inhibition assay):

Formulation # F1 F2 F3 SEC: % Monomer: % Monomer: % Monomer: start 99.198.9 99.1 1 month (5° C.) 98.5 98.6 98.6 3 months (5° C.) 99.1 98.9 99.06 months (5° C.) 98.7 98.3 98.7 HIC: % Unmodified: % Unmodified: %Unmodified: start 63 62 58 1 month (5° C.) 63 62 63 3 months (5° C.) 6060 60 6 months (5° C.) 59 61 62 % Specific % Specific Bioassay: %Specific Activity: Activity: Activity: start 105 107 100 1 month (5° C.)75 79 79 3 months (5° C.) 97 95 99 6 months (5° C.) 111 99 85

As shown by these results, the three liquid formulations have astability of at least 6 months at 5° C.

Example 12 Viscosity of Aqueous Solutions of E25 at High ConcentrationContaining Only Acetic Acid at Low Concentration

About 31 ml of the aqueous solution [127 mg/ml E25, 0.1% acetic acid]were prepared by ultrafiltration in a tangential-flow filtration system(membrane area: 150 cm², membrane cutoff: 10 kD, hold up volume of thesystem: 9 ml, retentate pressure: 2-3 bar), according to the three firststeps of the 5-steps procedure described in Example 7:

The starting solution was a solution of purified E25 antibody atconcentration 4.8 mg/ml in a 25 mM TRIS buffer pH 8 containing about 200mM NaCl. After pH adjustment to pH 5 with 5% acetic acid, the followingsteps were carried out:

-   -   In a first step, the solution was concentrated to 40 mg/ml.    -   In a second step, the concentrated solution was diafiltered at        constant retentate volume with 8 volumes of 0.1% acetic acid.    -   In a third step, the diafiltered solution was concentrated to        about 120 mg/ml and a sample was taken for viscosity        measurement.

The same experiment was carried out several times, changing only theacetic acid concentration used for the diafiltration.

The viscosity measurements were performed with a Paar Physica cone andplate rheometer at 23° C. and at a shear rate of 220 s⁻¹. The followingresults were obtained:

Acetic acid concentration E25 conc. Viscosity   0.1% (i.e. 17.3 mM) 127mg/ml 12.1 mPa · s   0.1% (i.e. 17.3 mM) 111 mg/ml  7.4 mPa · s  0.05%(i.e. 8.7 mM) 118 mg/ml  9.4 mPa · s  0.025% (i.e. 4.3 mM) 121 mg/ml13.8 mPa · s  0.01% (i.e. 1.7 mM) 121 mg/ml 17.8 mPa · s  0.005% (i.e.0.87 mM) 120 mg/ml 24.4 mPa · s 0.0025% (i.e. 0.43 mM) 115 mg/ml 25.4mPa · s  0.001% (i.e. 0.17 mM) 120 mg/ml 26.7 mPa · s    0% (i.e. wateralone) 116 mg/ml 47.2 mPa · s

As shown by these results, the beneficial effect of acetic acid comparedto water is already observed at an acetic concentration as low as 0.17mM, which allows to prepare an antibody solution at a concentration of120 mg/ml having a viscosity significantly lower than 50 mPa·s (i.e. thecorresponding viscosity obtained with water alone).

Example 13 Viscosity of Aqueous Solutions of E25 Containing Only 0.1%Acetic Acid, in Function of the Antibody Concentration

The same experiment as stated in Example 12 was repeated using 0.1%acetic acid for the diafiltration step, but this time the diafilteredsolution was concentrated to about 240 mg/ml (instead of 120 mg/ml).After recovery of the retentate out of the ultrafiltration system andfiltration through a 0.2 μm filter, a sample was taken for viscositymeasurement. Other samples were taken as well, for viscositymeasurements after various dilution steps with 0.1% acetic acid.

The viscosity measurements were performed with a Paar Physica cone andplate rheometer at 23° C. and at a shear rate of 220 s⁻¹. The followingresults were obtained:

Acetic acid conc. E25 conc. Viscosity 0.1% 240 mg/ml 225 mPa · s  0.1%220 mg/ml 125 mPa · s  0.1% 200 mg/ml 63 mPa · s 0.1% 180 mg/ml 40 mPa ·s 0.1% 170 mg/ml 35 mPa · s 0.1% 148 mg/ml 20 mPa · s 0.1% 127 mg/ml 12mPa · s 0.1%  85 mg/ml  6 mPa · s

As shown by these results, the beneficial effect of acetic acid allowsto prepare antibody solutions at a concentration up to about 180 mg/ml,having a viscosity significantly lower than 50 mPa·s.

Example 14 Use of Citric Acid as Acidic Component

About 19 ml of an aqueous solution of E25 at a concentration of about155 mg/ml in purified water having a pH of 4.4-4.6 adjusted with citricacid were prepared by ultrafiltration in a tangential-flow filtrationsystem (membrane area: 150 cm², membrane cutoff: 10 kD, hold up volumeof the system: 10 ml, retentate pressure: 2-3 bar), according to aprocedure similar to the 3-steps procedure described in Example 7:

The starting solution was a solution of purified E25 antibody atconcentration 4.8 mg/ml in a 25 mM TRIS buffer pH 8 containing about 200mM NaCl. After pH adjustment to pH 4.7 with 0.5 M citric acid,corresponding to a resulting citric acid concentration of about 6.6 mM,the following steps were carried out:

-   -   In a first step, H was tried to concentrate the solution to 40        mg/ml. But the filtrate flow decreased immediately very quickly,        so that it was not possible to carry out this concentration step        at the pH value of 4.7. In order to recover a normal filtrate        flow, the pH of the solution was lowered by stepwise addition of        small amounts of 0.5 M citric acid. Neither pH 4.4 nor pH 4.2        was low enough to allow a satisfactory filtrate flow. Finally,        the concentration step was possible only after pH lowering to pH        4.0, corresponding to a resulting citric acid concentration of        about 9 mM.    -   In a second step, the concentrated solution was diafiltered at        constant retentate volume with 8 volumes of purified water        having a pH of about 4.4, preliminarily adjusted with a few        droplets of 0.5 M citric acid, corresponding to a resulting        citric acid concentration in the range of about 0.05 to 0.1 mM.    -   In a third step, the diafiltered solution was concentrated as        high as possible. After recovery of the retentate out of the        ultrafiltration system and filtration through a 0.2 μm filter, a        sample was taken for concentration and pH measurements.

The maximal reachable concentration was 155 mg/ml, with a resulting pHof 4.5. In comparison, the maximal concentration obtained by using 0.1%acetic acid (without other additives) with the same ultrafiltrationequipment was about 240 mg/ml.

Moreover, a sample of this concentrated solution (155 mg/ml, pH 4.5) wastaken for addition of sodium citrate buffer of pH 4.5 to a foreseenfinal buffer concentration of 17.5 mM. But already after the addition ofthe first droplets (of 1 M sodium citrate buffer pH 4.5), E25 aggregatedimmediately and the solution became white turbid, turning soon into awhite solid gel. If 1 M MgCl₂ (instead of 1 M sodium citrate pH 4.5) isadded to the final concentrated solution of Example 14 (to a final MgCl₂concentration of 50 mM), the solution remains clear.

1. A stable aqueous solution comprising an E25 anti-IgE antibody at aconcentration of at least 80 mg/ml, and further comprising acetic acidat a concentration of at least 0.001%.
 2. A stable aqueous solutioncomprising an E25 anti-IgE antibody at a concentration of at least 80mg/ml and acetic acid, wherein the stable aqueous solution does notcontain sodium acetate.
 3. The stable aqueous solution of claim 1,wherein the acetic acid is present in a final concentration of at leastabout 0.01%.
 4. The stable aqueous solution of claim 1 wherein the pH ofsaid aqueous solution is above pH
 3. 5. The stable aqueous solution ofclaim 1 wherein the pH of said aqueous solution is between pH 3 andabout pH
 6. 6. The stable aqueous solution of claim 1 wherein less than5% of the E25 is present as aggregates after storage for at least 1month at 30° C. and/or at least 1 year at about 4° C.
 7. The stableaqueous solution of claim 1, further including CaCl₂.
 8. The stableaqueous solution of claim 1, further including MgCl₂.
 9. The stableaqueous solution of claim 1, further including at least one additive.10. The stable aqueous solution of claim 9, wherein the additive ispolysorbate.
 11. The stable aqueous solution of claim 9, wherein theadditive is a sugar.
 12. The stable aqueous solution of claim 11,wherein the sugar is selected from the group consisting of trehalose,sucrose, mannitol, sorbitol, fructose, maltose, lactose and dextran. 13.The stable aqueous solution of claim 9, wherein the additive is abuffering agent.
 14. The stable aqueous solution of claim 1 wherein saidaqueous solution is isotonic.
 15. A nasal spray comprising the stableaqueous solution as claimed in claim
 1. 16. A slow release formulationcomprising the stable aqueous solution as claimed in claim
 1. 17. Theslow release formulation of claim 16 selected from the group consistingof polymeric nanoparticles, polymeric microparticles, and gels.
 18. Theslow release formulation of claim 17, wherein the gel is a hyaluronicacid gel.
 19. A delivery system which contains the aqueous solution asclaimed in claim 1, selected from the group consisting of single useinjection syringes and inhalation devices.
 20. A process for thepreparation of an aqueous solution according to claim 1, which processcomprises admixing an E25 anti-IgE antibody with an acetic acid acidiccomponent.
 21. A process for the preparation of a therapeutic liquidformulation comprising an E25 anti-IgE antibody, wherein in a first stepan aqueous solution including an E25 anti-IgE antibody at aconcentration of at least 80 mg/ml and acetic acid at a concentration ofat least 0.01% is prepared, and, in a second step, at least onepharmaceutically acceptable additive is added to said aqueous solution.22. A process for the preparation of a therapeutic liquid formulationcomprising an E25 anti-IgE antibody at a concentration of more than 80mg/ml, wherein in a first step an E25 anti-IgE antibody solution in asuitable buffer is concentrated to a concentration of between about 10mg/ml and 80 mg/ml; in a second step, the concentrated solution obtainedin the first step is diafiltered with an aqueous solution of aceticacid, optionally containing MgCl₂ and/or CaCl₂ and/or further suitableadditives; and, in a third step, the solution obtained in the secondstep is further concentrated to a concentration of more than 80 mg/ml.23. A process for the preparation of a therapeutic liquid formulationcomprising an E25 anti-IgE antibody at a concentration of more than 80mg/ml, wherein in a first step an E25 anti-IgE antibody solution in asuitable buffer is concentrated to a concentration of between about 10mg/ml and 80 mg/ml; in a second step, the concentrated solution obtainedin the first step is diafiltered with an aqueous solution of aceticacid; in a third step, the solution obtained in the second step isfurther concentrated to an intermediate concentration of between about100 and 150 mg/ml E25 anti-IgE antibody; in a fourth step, theintermediate concentrated solution obtained in the third step isdiafiltered with an aqueous solution of acetic acid and furthercontaining MgCl₂ and/or CaCl₂ and/or further suitable additives; and, ina fifth step, the solution obtained in the fourth step is furtherconcentrated to a concentration of more than 150 mg/ml E25 anti-IgEantibody.
 24. The process of claim 23, wherein between the third andfourth step a solution of MgCl₂ and/or CaCl₂ and/or further suitableadditives is directly added to the intermediate concentrated solutionobtained in the third step.
 25. The process of claim 20, wherein theacidic component is present in a final concentration of at least 0.001%.26. The process of claim 20, wherein the pH of said aqueous solution isabove pH
 3. 27. The process of claim 20, wherein the pH of said aqueoussolution is between pH 3 and about pH
 6. 28. A therapeutic liquidformulation obtained by preparing an aqueous solution including an E25anti-IgE antibody at a concentration of at least 80 mg/ml and aceticacid at a concentration of at least 0.01%; and adding at least onepharmaceutically acceptable additive to said aqueous solution.
 29. Thetherapeutic liquid formulation of claim 28 wherein less than 5% of theE25 is present as aggregates after storage for at least 1 month at 30°C. and/or at least 1 year at about 4° C.
 30. The therapeutic liquidformulation of claim 28, further including CaCl₂ at a concentration ofbetween about 50 mM and 200 mM.
 31. The therapeutic liquid formulationof claim 28, further including MgCl₂ at a concentration of between about50 mM and 200 mM.
 32. The therapeutic liquid formulation of claim 28,further including at least one additive.
 33. The therapeutic liquidformulation of claim 32, wherein the additive is polysorbate.
 34. Thetherapeutic liquid formulation of claim 32, wherein the additive is asugar.
 35. The therapeutic liquid formulation of claim 34, wherein thesugar is selected from the group consisting of trehalose, sucrose,mannitol, sorbitol, fructose, maltose, lactose and dextran.
 36. Thetherapeutic liquid formulation of claim 32, wherein the additive is abuffering agent.
 37. The therapeutic liquid formulation of claim 28,wherein said liquid formulation is isotonic.
 38. A process for thepreparation of a therapeutic liquid formulation comprising an anti-IgEE25 antibody, which process comprises adding acetic acid on the lastpurification step of the preparation of said antibody.
 39. A stableaqueous solution, comprising: (i) an E25 anti-IgE antibody at aconcentration of at least 80 mg/ml, (ii) acetic acid and (iii) CaCl₂and/or MgCl₂.
 40. The stable aqueous solution of claim 39, wherein theconcentration of CaCl₂ is between about 50 mM and 200 mM.
 41. The stableaqueous solution of claim 39, wherein the concentration of MgCl₂ isbetween about 50 mM and 200 mM.
 42. The stable aqueous solution of claim39, wherein the acetic acid is present in a final concentration of atleast 0.001%.
 43. The stable aqueous solution of claim 39, wherein theacetic acid is present in a final concentration of at least 0.01%.
 44. Amethod of treating an allergic disease in a patient, comprisingadministering to the patient an effective amount of the stable aqueoussolution according to claim 39.